Identification and use of high efficacy vaccine antigens which modulate antigen presenting cells

ABSTRACT

The present invention relates to the identification and use of high efficacy vaccine antigens which modulate antigen presenting cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityof U.S. patent application Ser. No. 10/246,086 filed Sep. 16, 2002,which is a continuation of and claims the benefit of priority of U.S.patent application Ser. No. 09/313,487 filed May 17, 1999, now U.S. Pat.No. 6,680,176; both of which are hereby expressly incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of immunology. Morespecifically, novel biotechnological tools, therapeutics andphophylactics, which modulate antigen presenting cell activity aredisclosed.

BACKGROUND OF THE INVENTION

The mature, circulating, antigen specific cells of the immune systemface a challenge that does not trouble most other cells of the body.They must find each other, and they must do it often and quickly, everytime there is need of an immune response. The problem is compounded bythe rarity of the communicating partners, since only about 1 in 10⁴circulating B lymphocytes can react to any particular antigen and thefrequency of antigen specific T cells is thought to be similarly low.(Kennedy et al., J. Immuno. 96:973-980 (1966) and Vann, D. C. andDotson, D. R., J. Immunol. 112:1149-1157 (1974)). Thus, a rare T helpercell specific for a particular pathogen needs to find an equally rare Bcell specific for the same antigen. The fact that these encounters occurat all is due to the circulation patterns of these cells in lymph nodesand other specialized organs, as well as, the B cell's ability to act asan antigen presenting cell (APC) and attract the appropriate helper bycreating a surface display of MHC class II molecules loaded withpeptides from the antigen the B cell has captured. (Tony, H. P., andParker, D. C., J. Exp. Med. 161:223-241 (1985) and Lanzavecchia, A.,Nature 314:537-539 (1985)).

Once a B cell has attracted the right T helper cell, it uses a family ofreceptor-ligand pairs such as B7, CD40, and various cytokine receptorsto stimulate the T cell and receive stimuli in turn. (Foy, T. M. et al.,Semin. Immunol. 6:259-266 (1994)). T killer cells cannot do this and, ifthe two cell exchange between rare T and B cells seems challengingenough, the problem is far worse for communication between T helpers andT killer cells where the interaction requires a third participant, anAPC, that brings the T helpers and T killers together by displayingantigens to both. (Keene, J. A. & Forman, J., J. Exp. Med. 155:768-782(1982); Mitchison, N. A. & O'Malley, C., Eur. J. Immunol. 17:1579-1583(1987); and Bennett, S. R. et al., J. Exp. Med. 186:65-70 (1997)).

The problem is two fold. First there is the challenge of bringingtogether three rare circulating cells. Second, since T killers do notexpress the sorts of co-stimulatory molecules expressed by B cells andAPCs, (and, in mice, do not express MHC class II molecules with which topresent antigen to helper T cells) the question of how help isstimulated and delivered remains.

Currently, investigators believe that dendritic cells exist in only twostates: resting (an “immature” state) and activated (a “mature” state).In the activated state, a dendritic cell can present antigen andstimulate T helper cells, but not T killers. The successful priming ofkiller T cells is believed to require a three cell interaction betweenrare antigen loaded APCs and rare antigen specific helper T cells andkiller T cells. In the model set forth by Keene and Forman, for example,the presenting cell has a rather passive relationship with the killer Tcell and, like a B cell, the APC functions mainly to stimulate thehelper cell, which then secretes cytokines necessary for the growth andactivation of the neighboring killer T cell. (Keene, J. A. & Forman, J.,J. Exp. Med. 155:768-782 (1982)).

For several reasons this picture is not completely satisfying. First,there is no guarantee that a rare T helper and an equally rare T killershould find the same APC at the same time. Because resting killersrecognizing antigen become tolerant if there is no help available, manypotentially useful killers would be rendered useless by the lack ofimmediate help. (Guerder, S. & Matzinger, P., J. Exp. Med. 176:553-564(1992); Guerder, S. & Matzinger, P., Cold Spring Harb. Symp. Quant.Biol. 54:799 (1989); and Rees, M. A. et al., Proc. Natl. Acad. Sci.U.S.A. 87:2765-2769 (1990)). Second, the T helper would wastefullysecrete its cytokines into an environment that may contain no killers toreceive them. Third, killer responses to certain viruses are unimpairedby the absence of helper cells. (Tripp, R. A., et al., J. Immunol.155:2955-2959 (1995); Buller, R. M. et al., Nature 328:77-79 (1987);Cardin, R. D. et al., J. Exp. Med. 184:863-871 (1996); Hou, S. et al.,J. Virol. 69:1429-1434 (1995); Ahmed, R. et al. J. Virol. 62:2102-2106(1988); and Leist, T. P. et al., Scand. J. Immunol. 30:679-686 (1989)).The three cell interaction model offers no explanation for the existenceof these helper independent killer responses. In view of the foregoingand not withstanding the various efforts exemplified in the prior art,clearly several crucial pieces of the puzzle are missing.

SUMMARY OF THE INVENTION

In the present invention, we demonstrate that an APC, preferably adendritic cell, can be stimulated to a third state—a “superactivated”state. T helper cells, some viruses, and some antigens induce thedendritic cell to manifest the superactivated state. In contrast toactivated dendritic cells, superactivated dendritic cells have theability to activate a killer T cell by forming a two cell complex havingthe superactivated dendritic cell and the killer T cell. Notably, thesuperactivated APC activates a killer cell in the absence of a T helpercell. Additionally, we have discovered that specific agents whichinteract with the APC superactivate the APC or block, inhibit, orprevent the activation of killer T cells by interacting with the APC. Weshow that through modulation of the activation state of an APC, such asa dendritic cell by, for example, administering antibodies whichinteract with the APC, the activation of a T cell is concordantlygoverned.

In embodiments of the present invention, we reveal novelbiotechnological tools, prophylactics, therapeutics, and methods of useof the foregoing for modulating the activation state of an APC andthereby modulating the activation of a killer T cell. These embodimentshave several uses and applications in the field of immunology, andenable one of skill in the art to manufacture novel pharmaceuticals,therapeutic and prophylactic agents, and vaccine components for thetreatment and prevention of cancer, systemic infection, and autoimmuneresponses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates two models of the delivery of help to CD8 killers.Panel A depicts the “passive” model in which the dendritic cell presentsantigen to both the T helper and the killer but delivers co-stimulatorysignals only to the helper, which is thereby stimulated to produce IL-2for use by the nearby killer. Panel B depicts the “dynamic” model inwhich the dendritic cell offers co-stimulatory signals to both cells inthat it initially stimulates the T helper, which, in turn, stimulatesand “conditions” the dendritic cell to differentiate to a state where itcan now directly co-stimulate the killer.

FIG. 2 shows four ways to help a killer T cell. Set 1) Unseparated (a)or CD4 depleted (b, c) spleen cells from female B6 mice were stimulatedin vitro with B6 male spleen cells with or without CAS. Set 2) B6 femaleCD4 depleted spleen cells were stimulated in vitro with B6 maledendritic cells (dc) (d); dc+Marilyn (e); CD40 modulated dc (f); MHCclass II dc (g); CD40 modulated NMC II KO dc (h); MHC II KO dc infectedwith Influenza (i). Solid lines indicate the killing on male targets,whereas, dashed lines indicate female targets and diamonds representunprimed controls.

FIG. 3 shows a summary of 117 tests that demontrate five ways to help akiller T cell. Spleen cells from anti-H-Y primed female B6 mice weredepeleted or not of CD4 T cells and then stimulated with various normal(●) or MHC II KO (◯) male stimulators. Reading left to right: spleencells, dendritic cells (dc), dc incubated overnight with Marilyn, dcincubated overnight with Marilyn and then sorted to remove the T cells(+/−Marilyn), dc+ 10% CAS, dc modulated with a hamster (H) or a rat (R)anti-CD40 mAb, dc infected with influenza virus. Each point representsthe killing from a single culture at an R:T ratio at which killing shownby cultures from control mice drops off plateau. Background killing onfemale targets is subtracted. Horizontal lines are the group average.

FIG. 4 shows that B7.1 and B7.2 are involved in stimulation bycondtioned dendritic cells. Unseparated (expt.1) or CD4 depleted (expt.2) responders were stimulated respectively with CD40 modulated B6 or MHCII KO dc in the presence of titrated amounts of various blockingreagents.

FIG. 5 shows that virgin killer T cells can be primed in vivo by CD40modulated dendritic cells. A total of 173 B6 or MHC class II female micewere left untreated (♦) or injected once or twice with B6 (●) or MHC IIKO (◯) male spleen or dendritic cells that were untreated or modulatedwith hamster anti CD40 mAb. The in vitro cultures contained 10% CAS,which substitutes for help and allows us to concentrate on whether themouse was primed in vivo. All mice generated CTLs to third party CBA/Jtargets. Representation of killing activity is as in FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the present invention, we demonstrate that activation of a killer Tcell need not require the formation of a three-member complex having anAPC, a T helper cell, and a T killer cell. (See FIG. 1, Panel A).Rather, the activation of a killer T cell can occur in a two cellcomplex and two sequential steps. (See FIG. 1, Panel B). Accordingly, inthe first step, an APC stimulates a T helper T cell, which in turnstimulates or “superactivates” the APC to differentiate to a state whereit can independently stimulate a killer T cell. In the second step, theAPC encounters the killer T cell and stimulates it so that killer T cellpriming is achieved in a helper independent fashion. Furthermore, wehave discovered that the first step (“help”) can be bypassed altogetherby viral infection or an interaction with certain molecules at the cellsurface of APCs. Novel biotechnological tools, prophylactics,therapeutics, diagnostics, and methods of use of the foregoing formodulating the superactivation of an APC and thereby resulting in theactivation of a killer T cell are provided.

In the following disclosure several mechanisms are postulated todescribe how an APC is stimulated to a state capable of killer T cellactivation. These explanations are offered only to aid in theunderstanding of the field of the invention and consequently, theyshould be viewed as examples only and not limitations to embodiments ofthe present invention. Accordingly, the antigen specific T helper cellsmay stimulate the APC in a complex without a killer T cell andsuperactivated APCs can then prime the CD8 killer T cell. Rare,antigen-specific T helpers would then be able to make up for theirscarcity by inducing the differentiation of several APCs and therebyassist many CTL precursors without the need to simultaneously contactthe antigen. This model advantageously explains helper independentkiller responses to certain viruses if, for example, an infected APCresponds to the infection by directly undergoing superactivation.

In the discussion that follows, we disclose our discovery that help forkillers can be delivered through an APC without a T helpercell/APC/killer T cell complex. Notably, we have found that CD4⁺ Thelpers can route their activity through dendritic cells (labeled by theliterature as the best professional APCs). (Lassila, O. et al., Nature318:59-62 (1985); and Steinman, R. M., Annu. Rev. Immunol. 9:271-296(1991)). We demonstrate that activated dendritic cells cannot activatekillers, although they can present antigen and stimulate CD4+ T helpersto proliferate and produce cytokines. However, after an interaction witha helper T cell or antibodies to the surface molecule CD40 or viralinfection, activated dendritic cells differentiate into a superactivatedstate in which they are able to activate killer T cells in the absenceof any further need for T help.

Memory Killers are Helper Dependent

We chose to study the response to the male antigen H-Y for severalreasons. First, unlike killer T cells which respond to many viruses,killer T cells which respond to H-Y have long been known to be dependenton T helper cells. (Tripp, R. A., et al., J. Immunol. 155:2955-2959(1995); Buller, R. M. et al., Nature 328:77-79 (1987); Cardin, R. D. etal., J. Exp. Med. 184:863-871 (1996); Hou, S. et al., J. Virol.69:1429-1434 (1995); Ahmed, R. et al. J. Virol. 62:2102-2106 (1988);Leist, T. P. et al., Scand. J. Immunol. 30:679-686 (1989); Guerder, S. &Matzinger, P., J. Exp. Med. 176:553-564 (1992); Simpson, E. & Gordon, R.D., Immunol. Rev. 35:59-75 (1977); and von Boehmer, H. et al., Proc.Natl. Acad. Sci. U.S.A. 75:2439-2442 (2978)). Second, there are few, ifany, environmental antigens that cross react with H-Y. Third, T cellsfrom normal virgin female mice do not respond to H-Y in vitro unlessthey have first been primed in vivo, allowing us to differentiate easilybetween primary and secondary responses. (Simpson, E. & Gordon, R. D.,Immunol. Rev. 35:59-75 (1977) and Gray, D. & Matzinger, P., J. Exp. Med.174:969-974 (1991)).

FIG. 2 illustrates the helper dependence of the CD8 T killer responseand shows that T help can be delivered by soluble factors. FemaleC57Bl/6 (B6) mice, immunized in vivo with male spleen cells, generatedgood in vitro killer-cell responses against male spleen stimulators(FIG. 2 a-c). The response disappeared if we depleted CD4 helpers fromthe responding populations just before the in vitro culture andreappeared with the addition of a supernatant from Conconavalin-A(Con-A) stimulated T cells (CAS). These data document the helperdependence of the memory anti-H-Y response and lend some support toKeene and Forman's view that help can be delivered by soluble factors.(Keene, J. A. & Forman, J., J. Exp. Med. 155:768-782 (1982)).

The following discusses our finding that an interaction with CD40molecules of an APC activate killer T cells in the absence of T helpercells.

Help Can be Replaced Through CD40

Newborn female mice, which have only a minute number of anti male Tcells, and B6.bm12 mice, which carry a mutated MHC class II molecule,and are consequently deficient in H-Y specific T helpers, cannot beprimed against H-Y with an injection of male spleen cells, but bothtypes of mice respond quite well when primed with enriched, activateddendritic cells. (Matzinger, Annu. Rev. Immunol. 12:991-1045 (1994) andKatona et al., J. Immunol. 146: 4215-4221 (1991)). FIGS. 2 d-i show thatenriched and activated dendritic cells were very inefficient atstimulating CD8 killers from which the CD4 helper cells had beencompletely removed, and that the response was restored by the additionof Marilyn, a CD4⁺, H-Y specific, Th1 helper clone (FIG. 2 e). Thus, asmall number of helpers may go a long way, but without them, dendriticcells are unable to stimulate CD8 memory cells against H-Y.

Antibodies that were cross-linked to the surface molecule, CD40, werepreviously shown to stimulate B cell proliferation and enhance thefunction of dendritic cells to present antigen and stimulate T helpercells. (Saeland, et al., J. Exp. Med. 178: 113-120 (1993), Cella et al.,J. Exp. Med. 184: 747-752 (1996), and Yang and Wilson, Science273:1862-1864 (1996)). To determine whether treatment with anti-CD40 canbypass the need for T helpers in CD8 responses, we stimulated anenriched population of dendritic cells overnight by cross-linkingantibodies to CD40 and found that the dendritic cells gained the abilityto stimulate an anti-H-Y response (FIG. 2 f).

Next, we tested whether the result with the anti-CD40 treated dendriticcells was due to the stimulation of IL-2 production from contaminatingmemory CD4 helper T cells. By using dendritic cells from mutant MHCclass II Knock Out mice (KO), which have no MHC class II and cannotstimulate CD4 helper cells, we verified that enriched and activated KOdendritic cells, like their unmutated cousins, cannot stimulate killerresponses from CD4 depleted memory populations (FIG. 2 g). This resultwas not due to the lack of the H-Y antigen because the response isrestored by the addition of an IL-2 containing supernatant. Although theabsence of MHC class II molecules prevents KO dendritic cells fromdisplaying antigen to any contaminating T helper cells, they cannevertheless be superactivated, by overnight treatment with anti-CD40antibodies, to become excellent stimulators of a killing response (FIG.2 h). Control antibodies to dendritic cell surface antigens did not havethis effect, proving that the CD40 molecule, rather than Fc receptors orother non-specific changes, were responsible.

The results above demonstrate that the requirement for help can bebypassed in two different ways: (1) by adding supernatants containinggrowth factors for the CD8 cells or (2) by superactivating the dendriticcells through CD40, a likely molecule to be stimulated by CD4⁺ Thelpers. After such stimulation, the superactivated dendritic cellsthemselves gain the ability to activate CD8 killers without the need forfurther interaction with a helper T cell.

In the discussion below, we show that independent antigens superactivatedendritic cells which, in turn, activate killer T cells.

Stimulation by a T Helper Independent Antigen

There are several viruses known to elicit killer responses in theabsence of help. MHC class II KO or CD4 depleted mice, for example, makeundiminished responses to Sendai virus, Ectromelia virus, Herpes virusand diminished but still potent responses to LymphocyticChoriomeningitis and Influenza virus. (Tripp, R. A., et al., J. Immunol.155:2955-2959 (1995); Buller, R. M. et al., Nature 328:77-79 (1987);Cardin, R. D. et al., J. Exp. Med. 184:863-871 (1996); Hou, S. et al.,J. Virol. 69:1429-1434 (1995); Ahmed, R. et al. J. Virol. 62:2102-2106(1988); and Leist, T. P. et al., Scand. J. Immunol. 30:679-686 (1989)).We reasoned that these viruses infect dendritic cells and induce achange in differentiation state similar to that induced by T helpercells. Evidence of this was found when we infected enriched male MHCclass II KO dendritic cells with Influenza and used them to stimulate ananti-H-Y killing response from primed, CD4 depleted, B6 spleen cells.FIG. 2 i shows that the infected KO dendritic cells were as potent asthose stimulated with anti-CD40 in their ability to stimulate thekillers.

FIG. 3 shows a summary of the responses from 117 tests, demonstratingthe range of the normal, CD4 depleted, and reconstituted responses.Although there is a certain amount of variation, it is clear that memoryCD8 T killers are stimulated in vitro by normal B6 male (solid circles)and MHC class II KO male (empty circles) dendritic cells that have beensuperactivated by stimulation with CD4 T helpers, treatment withanti-CD40, or virus infection. The column labeled “dc+/−Marilyn” alsoshows that the T helper cells need not remain with the dendritic cells.Aliquots of dendritic cells that were cultured overnight with Marilynand then FACS sorted to remove the helpers were as stimulatory as thosefrom which the helper cells were not removed (labeled “dc+Marilyn”).Thus, a rare antigen-specific helper T cell need not communicatedirectly with the responding killers. It can delegate its function bysuperactivating a dendritic cell.

In the discussion below, we describe our discovery that agents can beadministered to APCs to inhibit or prevent the superactivated dendriticcells' ability to activate killer T cells.

Blocking Surface Co-Stimulatory Molecules

With B cells, unseparated spleen cells, and dendritic cells that developfrom stem cells in vitro, it has been found that CD40 crosslinking leadsto increased expression of several surface molecules involved instimulation and co-stimulation of T cells, however, by FACs analysis, wedid not see many of these reported changes, probably because ourenriched spleen derived dendritic cells are already highly activated.(Yong, J. L. et al., Immunity 2:239-248 (1995); Wu, Y. & Liu, Y., Curr.Biol. 4:499-505 (1994); and Caux, C. et al., J. Exp. Med. 180:1263-1272(1994)). We found that both the activated and the superactivateddendritic cells expressed very high and equivalent amounts of theadhesion/co-stimulatory molecules B7.1, B7.2, ICAM-1, and Heat StableAntigen, and low levels of IL-2 receptor, FAS and FAS-ligand. Incubationwith Marilyn resulted in increased expression of MHC class I and II, butneither anti-CD40 nor infection with influenza induced similar changes(though all three treatments caused minor changes in the expression ofCD40 and CD1d). It did not appear, therefore, that quantitative changesin these co-stimulatory molecules account for the qualitative changes wesaw in stimulatory capacity.

To determine whether the B7 molecules are involved in the stimulation ofkillers by superactivated dendritic cells, we included two differenttypes of blocking reagents in the cultures; antibodies to B7.1 and B7.2,as well as a soluble form of a receptor for these two molecules,recombinant mouse CTLA-4-Ig. (Linsley et al., Science, 257:792-795(1992)). FIG. 4 a shows that the activity of normal unseparated B6spleen cells, responding to B6 CD40-crosslinked dendritic cells (aculture where both helpers and killers respond to the superactivateddendritic cells), was blocked by both types of reagents, and not by thenon-activating control antibody, Ly5.2. In FIG. 4 b, we demonstrate thatthe same reagents block the activity of CD4 depleted spleen cellsstimulated with CD40 cross-linked MHC class II KO dendritic cells. Fromthese results we conclude that, though the APC surface co-stimulatorymolecules involved in the activation of T helper cells are notsufficient for the stimulation of killers, they are neverthelessimportant. Interestingly, neither anti-B7.1 nor anti-B7.2 were able toblock alone, even at very high doses, while the two together blockedvery well, proving that T killers are able to use the two co-stimulatorymolecules interchangeably.

In addition to co-stimulatory molecules, dendritic cells can elaboratesoluble factors, and CD40 stimulated dendritic cells have been shown toproduce 1L-12. (Cella, M. et al., J. Exp. Med, 184:747-752 (1996) andKoch, F. et al., J. Exp. Med. 184:741-746 (1996)). We therefore soughtto bypass the need for help by adding IL-12 to the cultures but, unlikeCAS, IL-12 did not replace the activity of helpers. The most likelyscenario is that co-stimulation for a CD8, perhaps by an undiscoveredco-stimulatory molecule, is similar to co-stimulation for a CD4 in thatit results in the production of IL-2. (Mueller, D. L. et al., Ann. Rev.Immunol. 7:445-480 (1989) and Paliard, X. et al., J. Immunol.141:849-855 (1988)). In support of this, we found that CD4 depletedcells produced a small amount of IL-2 when stimulated by superactivatedMHC class II KO dendritic cells, whereas they produced no IL-2 whenstimulated by dendritic cells cultured without a superactivatingstimulus. This would explain why we can bypass the helper cell in twoways: first, by supplying the necessary IL-2 or second, bysuperactivating the dendritic cell to induce the CD8 killer to make itsown.

We describe below several experiments in which we demonstrate thatkiller T cells can be stimulated in vivo.

Priming Naive T Killer Cells in Vivo

There were several reasons to see whether our in vitro finding withmemory killer cells could be extended to naive killers in vivo. First,naive T cells are widely thought to have more stringent activationrequirements than memory T cells. (Fuchs, E. J. & Matzinger, P., Science158:1156-1159 (1992); Liu, Y. et al., J. Exp. Med. 185:251-262 (1997);and Croft, M. et al., J. Immunol. 152:2675-2685 (1997)). Perhaps naivekillers need a direct interaction with a helper T cell whereas memorycells do not. (Inaba, K. et al., J. Exp. Med. 166:182-194 (1987)).Second, in vitro and in vivo conditions do not always follow the samerules. For example, T cell populations deprived of helpers specific forsheep red blood cells are able to help for specific antibody responsesin vitro but not in vivo and, conversely, cross priming for killers canoccur in vivo but not in vitro. (Feeney, A. J. et al., J. Mol. CellImmunol. 1:211-222 (1984) and Bevan, M. J., J. Exp. Med. 143:1283-1288(1976)). Third, Mitchison's cluster model hinted that helper dependenceand independence may be determined by the frequency of responders.(Michison, N. A. & O'Malley, C., Eur. J. Immunol. 17:1579-1583 (1987)).If the frequency is high, the response may not depend on help. Thus, asthe reasoning goes, killer responses to viruses may be less dependent onhelp than those to H-Y simply because the frequency of responding cellsis higher.

We examined the primary anti-H-Y response of naive killers in MHC classII KO mice. As mentioned above, MHC class II KO mice are able to respondto various viruses and we found, as expected, that they would notrespond to male cells. (Tripp, R. A. et al., J. Immunol. 155:2955-2959(1995) and Hou, S. et al., J. Virol. 69:1429-1434 (1995). FIG. 5 showsthat normal B6 females were primed either with male spleen or maledendritic cells whereas the KO mice were unresponsive to both. Becauseof the possibility that the lack of response was due to a lack of helpin the in vitro culture, rather than a lack of help during priming invivo, we also tested these cells in cultures to which we added anexogenous source of helper factors. In neither case did the killers fromthe KO mice respond to male stimulators. Thus, though activated maledendritic cells can prime virgin bm12 mice (where help is limited), theycannot, by themselves, prime anti-H-Y killers in the MHC class II KOmice, where help is absent. (Boog, C. J., Nature 318:59-62 (1985)).

Next, we tested whether stimulation with anti-CD40 turns a dendriticcell into a superactivated APC that is able to prime naive T killercells. Surprisingly, it does. FIG. 5 is a summary of 173 female MHCclass II KO or B6 mice that were primed in vivo with male spleen cells,cultured activated dendritic cells, or anti-CD40 treated superactivateddendritic cells, and then restimulated in vitro in the presence ofsupernatants containing soluble helper factors. By replacing the needfor help in the in vitro culture, we were able to concentrate on the invivo priming step, since only primed killers can respond in vitro. Wefound that the KO mice were able to respond well only if they had beenprimed with anti-CD40 treated dendritic cells, and then only if theywere immunized twice. Thus, the helper requirement for virgin T killercells, like that for memory T killer cells, can be bypassed bysuperactivated APCs.

The requirement for repeated immunization is intriguing. It may be thatsuperactivated dendritic cells do not home as efficiently to lymph nodesas untreated cells, perhaps because of antibody remaining on theirsurface, or perhaps because they have differentiated to a state inwhich, under normal circumstances, they would have already migrated tothe lymph node and would not be required to wander further.Alternatively, the reason may have to do with time rather thangeography. An encounter between killer and dendritic cells may take acouple of days in vivo whereas it only takes a couple of hours in vitro.If the superactivated state is not eternally stable, many of theinjected dendritic cells may have reverted (or died) before they can beseen by a killer. The superactivated state may last a couple of days. Inmany experiments, we saw that normally cultured dendritic cells wereable to stimulate weakly. We surmise that this may be due to thepresence of a small percentage of dendritic cells that had beensuperactivated in vivo by T helper cells responding to environmentalantigens and that maintained their stimulatory ability during the twoday preparatory cultures.

There are several systems in which two cells communicate through athird. In every case but that of the T helper with its corresponding Tkiller, the interactions are either mediated by soluble factors thattraverse the distances required (such as the regulatory feedbackmechanisms in the hypothalamo-pituitary-adrenal axis) or are facilitatedby stable geometries in which the cells involved remain linked for theirentire lives (as in the nervous system). Neither of these mechanismsworks for cells of the immune system, where the communicating cells arerare and migratory. The two-cell interactions between antigen specific Thelpers and their corresponding B cells are facilitated by structures inlymph nodes, to which local tissue fluids drain and where the twocirculating cells can find each other during an infection. (Anderson, A.O. & Shaw, S., Semin. Immunol. 5:271-282 (1993)). However, faced with analmost impossible interaction of three migratory cells, two of which arerare (the T helper and killer) and one of which is both rare andtransient (the antigen loaded APC), the immune system adds the fourthdimension, time, and transforms the three cell interaction into a seriesof consecutive two cell engagements. The first, between a T helper andan activated antigen presenting cell, induces the APC to differentiateto a superactivated state in which it can engage in the second, andstimulate a T killer cell.

Mediating the help function through superactivated APCs solves more thanjust the rarity problem. Helpers need not excessively secrete IL-2 intotheir surroundings. Killers do not need to wait for a co-stimulatorysignal or help after binding to antigen, since both the antigen and theco-stimulatory signals come from APCs and can be deliveredsimultaneously, as they are to helpers. Furthermore, the activity of afew helpers is amplified, since a single T helper cell can “arm” anumber of APCs which can then, in their turn, activate a multitude ofkillers.

Superactivation of APCs also provides an explanation for thecontradictions about the helper independence of CTL. Dendritic cells,like many other cells, react directly to a virus infection. Whereaskeratinocytes, for example, respond to virus by producing interferon,dendritic cells respond by becoming superactivated and able to activatekiller T cells. (Torseth, J. W. et al., J. Infect. Dis. 155:641-648(1987)). Our discovery explains the helper independent responses tovirus in the class II KO mice. It also explains why some anti-viralresponses depend more heavily than others on T helpers. For example, theKO mice respond as well as wild type mice to Sendai virus but less wellthan wild type mice to Influenza virus. For these viruses, we believethat two types of dendritic cells present viral antigens to CD8 killers.There are those that are infected and consequently superactivated andare able to activate killers in the absence of help. However, there areothers that have become activated by danger signals at the site ofinfection but which are not themselves infected. (Matzinger, P., Annu.Rev. Immunol. 12:991-1045 (1994)). Though they will have picked up theviral antigens from dead and dying infected cells in their surroundings,they cannot become superactivated in the absence of help and, thus, in aKO mouse, will be unable to stimulate CD8 killers. A corollary is thatthe helper independence of a CD8 response to a particular viruscorrelates directly with the virus's ability to infect and superactivateAPCs.

As seen in FIGS. 2 i and 3, superactivation by viruses also substitutesfor help in responses directed at non-viral antigens that are presentedby the same APC. This finding explains why the subject of help forkillers has been fraught with contradictions. For example, in an earlierstudy with responses to Qa-1, we found that the killer responses wereindependent of help during a mouse hepatitis virus infection in theanimal colony. (Guerder, S. & Matzinger, P., J. Exp. Med. 186:553-564(1992)). In the current study, for a period of about four months, a lowbut nevertheless substantial response to H-Y remained in CD4 depletedCD8 memory cells. This helper “independent” aspect of the responsedisappeared when we purchased mice from another source colony (though noknown pathogen was found in the first colony). Thus, depending on thehealth status of a mouse colony, various responses appear to bedependent on or independent of T help. (Keene, J. A. & Forman, J., J.Exp. Med. 155:768-782 (1982); Guerder, S. & Matzinger, P., J. Exp. Med.186:553-564 (1992); Guerder, S. & Matzinger, P., Cold Spring Harb. Symp.Quant. Biol. 54:799 (1989); Rees, M. A. et al., Proc. Natl. Acad. Sci.U.S.A. 87:2765-2769 (1990); Simpson, E. & Gordon, R. D., Immunol. Rev.35:59-75 (1977); von Boehmer, H. et al., Proc. Nat. Acad. Sci. U.S.A.75:2439-2442 (1978); Fuchs, E. J. & Matzinger, P., Science 258:1156-1159(1992); Auchincloss, H., Jr. et al. Proc. Natl. Acad. Sci. U.S.A.90:3373-3377 (1993); Boog, C. J. et al., Nature 318:59-62 (1985);Shimuzu, T. & Takeda, S., Eur. J. Immunol. 27:500-508 (1997); Roopenian,D. C. et al., J. Immunol. 130:542-545 (1983); and Rosenberg, A. S. etal., Nature 322:829-831 (1986)).

Finally, the existence of superactivated dendritic cells makes it clearthat there are several activation states for dendritic cells, in analogyto the multiplicity of activation states seen with other cells of theimmune system. In the case of B cells, for example, signals from Thelper cells do more than simply evoke activation signals. They alsopersuade B cells to switch to the production of different classes ofantibody. Thus, Th1 helper T cells induce the production of thecomplement fixing antibody, IgG2a, whereas Th2 helpers elicit IgE andIgG1, and Th3 helpers signal the production of IgA. (Katona et al., J.Immunol. 146:4215-4221 (1991), Chen et al., Science 265:1237-1240(1994), and Stavnezer, J. Immunol. 155:1647-1651 (1995)). Similarly,dendritic cells are influenced by the signals they receive todifferentiate to different states of superactivation, each one involvedin a different class of response. We have shown here that dendriticcells cultured with Th1 helpers or virus become adept at activating CD8killers. There is also some evidence that the presence of IL-10 or fluidfrom the anterior chamber of the eye can prompt APCs to become goodinducers of TH2 rather than Th1 responses. (Liu, L. et al., Adv. Exp.Med. Biol. 417:375-381 (1997) and Wilbanks, G. A. & Streilien, J. W.,Eur. J. Immunol. 22:1031-1036 (1992)). The dendritic cell is a cell typewhich responds to its environment in several ways and, in turn,influences several aspects of an immune response. First, it is activatedby exogenous or endogenous danger signals to capture, process, andpresent antigen along with co-stimulatory signals and thus initiate animmune response. (Janeway, C. A., Jr., Cold Spring Harb. Symp. Quant.Biol. 54 Pt 1:1-13 (1989) and Matzinger, P., Annu Rev. Immunol.12:991-1045 (1994)). Further, it is also influenced by the cells,cytokines, and other signals in its environment to modify the responsethat it initiates, so that the response is appropriate for both thepathogen it is directed against and the location in which it unfolds.

In the discussion below, we describe several methods of molecularmodeling and rational drug design for the identification of more ligandswhich superactivate an APC or block, inhibit, or prevent the activationof killer T cells.

Methods of Rational Drug Design

Combinatorial chemistry is the science of synthesizing and testingcompounds for bioactivity en masse, instead of one by one, the aim beingto discover drugs and materials more quickly and inexpensively than wasformerly possible. In some embodiments, search programs are employed tocompare regions of ligands which superactivate an APC or inhibit thesuperactivation of killer T cells with other molecules, such aspeptides, peptidomimetics, and chemicals, so that therapeuticinteractions of the molecules can be predicted and new derivativeligands can be designed. (Schneider, Genetic Engineering News December:page 20 (1998), Tempczyk et al., Molecular Simulations Inc. SolutionsApril (1997), and Butenhof, Molecular Simulations Inc. Case Notes(August 1998)). This process of directed combinatorial chemistry isreferred to as “rational drug design”. One goal of rational drug designis to produce structural analogs of biologically active polypeptides ofinterest or of small molecules with which they interact (e.g., agonists,antagonists, null compounds) in order to fashion drugs which are, forexample, more or less potent forms of the ligand. (See, e.g., Hodgson,Bio. Technology 9:19-21 (1991)). Rational drug design has been used todevelop HIV protease inhibitors and agonists for five differentsomatostatin receptor subtypes. (Erickson et al., Science 249:527-533(1990) and Berk et al., Science 282:737 (1998)).

In one approach, a three-dimensional structure of a ligand of interest(e.g., a polypeptide or fragment corresponding to a ligand whichinteracts with CD40 or a ligand for CD40) is determined by x-raycrystallography, NMR, or neutron diffraction and computer modeling.Useful protein models of the ligand are also gained by computer modelingalone. Combinatorial chemistry is then employed to design derivatives ofthe ligand of interest based on the three-dimensional models. The APCsuperactivation, killer T cell activation, and superactivation blockingassays, described above and in Ridge et al., Nature 393:474 (1998)(referred to collectively as “superactivation assays”) are performed onthe derivative ligands and classes of ligands based on the potency ofAPC superactivation and killer T cell activation are recorded on acomputer readable media. Further cycles of modeling and superactivationassays are employed to more narrowly define the parameters needed in aligand which elicits a desired response.

For example, chemical libraries and databases are first searched formolecules similar in structure to one or more ligands which interactwith the APC at a specific site and produce a desired APC response(e.g., anti-CD40 antibodies—FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5,and M3 or other antibodies—anti-B7.1 (17A10), anti-B7.2 (2D10),CTLA-4-Ig, and MR1 and antibodies which recognize a CD40 ligand).Identified candidate ligands are then screened in the superactivationassays, described above, and the compounds which produce the desiredsuperactivation state are used as templates for further libraryconstruction. Libraries of derivative ligands are synthesized on solidsupport beads by split-and-pool synthesis, a multistage process forproducing very large numbers of compounds. The support-bound compoundsare then used in superactivation assays or “free mixtures” are createdby cleaving the compound from the support and these free mixtures arescreened in the superactivation assays. Compounds which producedesirable APC responses are identified, recorded on a computer readablemedia, and the process is repeated to select for optimal ligands.

Each compound and its response in a superactivation assay is recorded ona computer readable media and a database or library of compounds andrespective APC responses is generated. These databases or libraries areused by researchers to identify important property differences betweenactive and inactive molecules so that compound libraries are enrichedfor ligands which have favorable characteristics. Further, enrichmentcan be achieved by using approaches in dynamic combinatorial chemistry.(See e.g., Angnew, Chem. Int. Ed., 37:2828 (1998)). For example, an APCtarget biomolecule, such as CD40, is joined to a support and is bound bythe compounds from the libraries generated above. The CD40 resin boundwith one or more candidate ligands is removed from the binding reaction,the ligands are eluted from the support, and are identified. Cycles ofimmobilized target binding assays are conducted, classes of ligandswhich exhibit desired binding characteristics are identified, and thisdata is recorded on a computer readable media and is used to select moreligands which produce a desired APC response.

In addition, a ligand peptide of interest (e.g., anti-CD40antibodies—FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, and M3 or otherantibodies—anti-B7.1 (17A10), anti-B7.2 (2D10), CTLA-4-Ig, and MR1 andantibodies which recognize CD40 ligands) is analyzed by an alanine scan(Wells, Methods in Enzymol. 202:390-411 (1991)). In this technique, anamino acid residue is replaced by alanine, and its affect on thepeptide's activity is measured by a functional assay, such as thesuperactivation assays described herein. Each of the amino acid residuesof the peptide is analyzed in this manner and the important regions ofthe peptide are identified. Subsequently, this functionally importantregion is recorded on a computer readable medium, stored in a firstdatabase in a computer system, and a search program is employed togenerate a protein model of the functionally important region. Once aprotein model of the functionally important region has been generated, asecond database comprising one or more libraries having peptides,chemicals, peptidomimetics and other agents is accessed by a searchprogram and individual agents are compared to the protein model toidentify agents which comprise homologous regions or domains whichresemble the identified functionally important region. Agents identifiedby the approach above are then tested in the superactivation assays andare used to construct multimeric agents and/or are incorporated intopharmaceuticals, as detailed below.

In another embodiment, computer modeling and thesequence-to-structure-to-function paradigm is exploited to identifysuperactivating ligands or ligands which inhibit the superactivation ofkiller T cells. By this approach, first the structure of a proteinligand having a known response in a superactivation assay is determinedfrom its sequence using a threading algorithm, which aligns the sequenceto the best matching structure in a structural database. Next, theprotein's active site (i.e., the site important for a desired responsein the superactivation assay) is identified and a “fuzzy functionalform” (FFF)—a three-dimensional descriptor of the active site of aprotein—is created. (See e.g., Fetrow et al., J. Mol. Biol. 282:703-711(1998) and Fetrow and Skolnick, J. Mol. Biol. 281: 949-968 (1998)).

The FFFs are built by itteratively superimposing the protein geometriesfrom a series of functionally related proteins with known structures.The FFFs are not overly specific, however, and the degree to which thedescriptors is relaxed is explored. In essence, conserved andfunctionally important residues for a desired T cell response areidentified and a set of geometric and conformational constraints for aspecific function are defined in the form of a computer algorithm. Theprogram then searches experimentally determined protein structures froma protein structural database for sets of residues that satisfy thespecified constraints.

By using this computational protocol, genome sequence data bases such asmaintained by various organizations are screened for specific proteinactive sites and for identification of the residues at those activesites which resemble a desired ligand. Databases of short sequencepatterns or motifs designed to identify a given function or activity arealso screened. Several other groups have developed such databases. Thesedatabases, notably Prosite, Blocks, and Prints use short stretches ofsequence information to identify sequence patterns that are specific fora given function; thus they avoid the problems arising from thenecessity of matching entire sequences. By these approaches, new ligandsare rationally selected for further identification by thesuperactivation assay methods, described above. Rounds or cycles ofsuperactivation assays on the molecules and derivatives thereof andfurther FFF refinement and database searching allows us to more narrowlydefine classes of ligands which produce desirable killer T cellresponses.

Many computer programs and databases are used with embodiments of theinvention to identify agents which modulate superactivation of an APC.The following list is intended not to limit the invention but to provideguidance to programs and databases which are useful with the approachesdiscussed above. The programs and databases include, but are not limitedto: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group),GeneMine (Molecular Applications Group), Look (Molecular ApplicationsGroup), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI),BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403 (1990)), FASTA(Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988)),Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHAR (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), Modeller 4 (Sali and Blundell J. Mol. Biol. 234:217-241 (1997)),ISIS (Molecular Simulations Inc.), Quanta/Protein Design (MolecularSimulations Inc.), WebLab (Molecular Simulations Inc.), WebLab DiversityExplorer (Molecular Simulations Inc.), Gene Explorer (MolecularSimulations Inc.), SeqFold (Molecular Simulations Inc.), theEMBL/Swissprotein database, the MDL Available Chemicals Directorydatabase, the MDL Drug Data Report data base, the ComprehensiveMedicinal Chemistry database, Derwents's World Drug Index database, andthe BioByteMasterFile database. Many other programs and data bases wouldbe apparent to one of skill in the art given the present disclosure.

In the discussion that follows, several superactivation assays aredescribed for the determination of a ligand's capacity to superactivatean APC or block, inhibit, or prevent superactivation of an APC andthereby modulate killer T cell activation.

Identification of Ligands Which Modulate Superactivation of an APC

In the experiments presented above, we demonstrated that antibodies toCD40 and influenza virus superactivate an APC, a dendritic cell, andthat antibodies to the B7.1 and B7.2 molecules block the ability of APCsto activate a killer T cell. The term “superactivation” refers to an APCactivation state which allows for the activation of killer T cells at alevel above that exhibited by an unstimulated APC. The activation stateof an APC can be expressed in terms of the percentage killing of apathogen, for example. That is, the capacity of killer T cells to kill apathogen having an antigen in the absence of helper T cells which ispresented by an APC is directly related to the activation state of anAPC. A simply “active” APC exhibits a percentage killing on the order of0-10%, whereas, a “superactivated” APC desirably exhibits a percentagekilling between 20% and 100% and preferably between 30% and 100%. Asuperactivated APC, for example, exhibits a percentage killing of atleast 20% or 22% or 24% or 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%,44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%,72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, orabout 100%. Superactivation and the activation state of an APC can bemeasured by more direct biochemical approaches as will be apparent toone of skill in the art given this disclolsure.

Candidate ligands, ligands selected by the rational drug designapproaches discussed above, for example, and randomly selected ligandsare, desirably, screened by several “superactivation assays” so as todetermine the candidates ability to superactivate an APC or inhibit Tcell activation. One type of superactivation assay, as described above,contacts an APC, such as a dendritic cell, with a ligand in the absenceof T helper cells and the ligand-stimulated APC is then contacted withkiller T cells. Subsequently, the killer T cells are analyzed for theirability to kill a pathogen having the antigen presented by the APC andthe percentage of killing—a measure of the activation state of theAPC—is recorded on a computer readable media. Other ways of measuringthe activation state of an APC or a killer T cell, such as bybiochemical approaches, are within the scope of embodiments of thepresent invention and are encompassed with the meaning of the term“superactivation assay.”

Libraries of information on ligands and their correspondingsuperactivation state measurements for specific APCs and pathogensand/or specific antigens are generated by performing the superactivationassays described above and a record of the results is generated andstored on a computer readable media. Databases of this information isvaluable to investigators and clinicians for selecting the type ofligand-based pharmaceutical to treat a particular disease. Preferablelibraries are created by performing the superactivation assays above onthe ligands FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, M3 17A10, 2D10,CTLA-4-Ig, and MR1 and antibodies which recognize CD40 ligands andfragments or derivatives therof. These libraries are also used to mapthe active regions of the ligands and new ligands which produce adesired killer T cell response are identified using this information andthe methods presented above.

The immune responsiveness of a subject to a ligand is also predictedusing the assays detailed above. Accordingly, an APC provided from asubject is contacted with a ligand in the absence of T helper cells andthe activation state of the APC is determined. The immune responsivenessof the subject is then measured by determining the relative level ofactivation of the APC, wherein an activation state exhibiting a killingpercenatge of 20% to 100% indicates a good immune responsiveness and anactivation state exhibiting a killing percentage of 0% up to 20%indicates a poor immune responsiveness. A good immune responsiveness,for example, is indicated by superactivated APCs which exhibit a killingpercentage of at least 20% or 22% or 24% or 26%, 28%, 30%, 32%, 34%,36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%,64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%,92%, 94%, 96%, 98%, or about 100%. In preferable embodiments, the ligandis selected from FGK45, HM40-3, 5C3, Mab-89, BE-1, EA5, M3, 3/23, 17A10,2D10, CTLA-4-Ig, and MR1 and antibodies which recognize CD40 ligands andfragments or derivatives therof. A preferable APC is a dendritic cell.By using this approach to determine the immune responsiveness of asubject to a particular antigen present in a cancer cell or pathogen,for example, ligands which provide the best APC and killer T cellresponse for the particular antigen are identified and these ligands areselected for incorporation into pharmaceuticals.

A kit to identify immune responsiveness is another embodiment. One suchkit comprises an APC (preferably a dendritic cell), a ligand that isrecognizable by the APC (preferably one or more of the antibodies orfragments or derivatives of FGK45, HM40-3, 3/23, 17A10, 5C3, Mab-89,BE-1, EA5, M3, 2D10, CTLA-4-Ig, and MR1 and antibodies which recognizeCD40 ligands), a means for contacting the APC with the ligand, a meansto analyze the activation state of the APC, and a means to measure theimmune responsiveness, wherein superactivation indicates a good immuneresponsiveness and an activation state below superactivation indicates apoor immune responsiveness. Several means for contacting the APC with aligand are used with this embodiment and the bound-support agents andthe pharmaceutical preparations, described below, are examples. Theactivation state of the APC is frequently measured indirectly bymonitoring the percentage killing by killer T cells, as describedpreviously, or by biochemical assays as known to those of skill in theart. The means to measure immune responsiveness often times depends onthe means used to measure the activation state of the APC but generallyincludes the detection of markers such as radioactivity, fluorescence,magnetism, including automated detection approaches as known in the art.

In the dislcosure below, we teach the preparation of multimeric supportshaving ligands which modulate APC superactivation and thereby modulatekiller T cell activity. These multimeric supports have many usesincluding, but not limited to, the manufacture of biotechnological toolsand components for pharmaceuticals, therapeutic and prophylactic agents.

Preparation of Multimeric Supports and Multimerized Ligands

A useful biotechnological tool or a component to a prophylactic ortherapeutic agent provides a ligand in such a form or in such a way thata sufficient affinity or superactivation of the APC orinhibition/prevention of superactivation of the APC is obtained. While anatural monomeric ligand (i.e. appearing as a discrete unit carryingonly one binding epitope) is sufficient to superactivate an APC orinhibit superactivation of an APC, a synthetic ligand or a multimericligand (i.e. appearing as a multiple unit of the compound having severalbinding epitopes) often times has greater ability to superactivate anAPC or inhibit superactivation of an APC. It should be noted that theterm “multimeric” refers to the presence of more than one unit of aligand, for example, several individual molecules of an antibody, asdistinguished from the term “multimerized” which refers to the presenceof more than one ligand joined as a single discrete unit, for exampleseveral antibody molecules joined in tandem.

A multimeric agent (synthetic or natural) which modulates thesuperactivation of an APC is obtained by coupling a ligand to amacromolecular support. A “support” is also termed a carrier, a resin orany macromolecular structure used to attach or immobilize a ligand.Solid supports include, but are not limited to, the walls of wells of areaction tray, test tubes, polystyrene beads, magnetic beads,nitrocellulose strips, membranes, microparticles such as latexparticles, sheep (or other animal) red blood cells, Duracyte® artificialcells, and others. In several emodiments, the macromolecular support hasa hydrophobic surface which interacts with a portion of the ligand by ahydrophobic non-covalent interaction. In some cases, the hydrophobicsurface of the support is a polymer such as plastic or any other polymerin which hydrophobic groups have been linked such as polystyrene,polyethylene or polyvinyl. Additionally, the ligand is covalently boundto carriers including proteins and oligo/polysaccarides (e.g. cellulose,starch, glycogen, chitosane or aminated sepharose). In these laterembodiments, a reactive group on ligand, such as a hydroxy or an aminogroup, is used to join to a reactive group on the carrier so as tocreate the covalent bond. Embodiments also comprise a support with acharged surface which interacts with the ligand. Additional embodimentscomprise a support which has other reactive groups that are chemicallyactivated so as to attach a ligand, such as a peptide or chemicalcompound. For example, cyanogen bromide activated matrices, epoxyactivated matrices, thio and thiopropyl gels, nitrophenyl chloroformateand N-hydroxy succinimide chlorformate linkages, or oxirane acrylicsupports are used. (Sigma).

Inorganic carriers, such as silicon oxide material (e.g. silica gel,zeolite, diatomaceous earth or aminated glass) to which the ligand iscovalently linked through a hydroxy, carboxy or amino group and areactive group on the carrier are also embodiments. Furthermore, in someaspects, a liposome or lipid bilayer (natural or synthetic) is used as asupport and ligands are attached to the membrane surface or areincorporated into the membrane by techniques in liposome engineering. Byone approach, liposome multimeric supports comprise fusion proteinshaving a first domain which interacts with a molecule on the surface ofan APC and a second domain which anchors the protein to the lipidbilayer. The anchor is constructed of hydrophobic amino acid residues,resembling known transmembrane domains, or comprises ceramides that areattached to the first domain by conventional techniques.

Carriers for use in the body, (i.e. for prophylactic or therapeuticapplications) are desirably physiological, non-toxic and preferably,non-immunoresponsive. Contemplated carriers for use in the body includepoly-L-lysine, poly-D, L-alanine and Chromosorb® (Johns-ManvilleProducts, Denver Co.). Ligand-conjugated Chromosorb® (Synsorb-Pk) hasbeen tested in humans for the prevention of hemolytic-uremic syndromeand was reported as not presenting adverse reactions. (Armstrong et al.J. Infectious Diseases 171:1042-1045 (1995)). For some embodiments, theadministration of a “naked” carrier (i.e., lacking an attached ligand)which has the capacity to attach a ligand which modulates APCsuperactivation and killer T cell activation inside the body of asubject is performed. By this approach, a “prodrug-type” therapy isadministered in which the naked carrier is provided separately from thedesired ligand and, once both are in the body, the carrier and theligand assemble into a multimeric complex and superactivate an APC.

In another embodiment, linkers, such as 8 linkers, of an appropriatelength are inserted between the ligand and the support so as toencourage greater flexibility in the ligand and thereby overcome anysteric hindrance which is presented by the support. The determination ofan appropriate length of linker which allows for optimal binding andsuperactivation of the APC or inhibition of superactivation of the APC,is made by screening the ligands with varying linkers in thesuperactivation assays detailed in the present disclosure.

A composite support comprising more than one type of ligand is also anembodiment. A “composite support” is a carrier, a resin, or anymacromolecular structure used to attach or immobilize two or moredifferent ligands which modulate the superactivation of an APC. In someembodiments, a liposome or lipid bilayer (natural or synthetic) is usedin constructing a composite support and ligands are attached to themembrane surface or are incorporated into the membrane using techniquesin liposome engineering. By one approach, a liposome composite supportcomprises fusion proteins having a first domain which interacts with amolecule on the surface of an APC and a second domain which anchors theprotein to the lipid bilayer. The anchor is constructed of hydrophobicamino acid residues, resembling known transmembrane domains, orcomprises ceramides that are attached to the first domain byconventional techniques Many different fusion proteins are created inthis manner and are incorporated into a liposome so as to create thevarious aspects of composite supports of the present invention. Thecomposite supports are also constructed by utilizing hydrophobicinteractions and covalent linkages formed through reactive groups, asdetailed above.

Linkers, such as 8 linkers, of an appropriate length between the ligandsand the support are inserted in some embodiments so as to encouragegreater flexibility in the molecule and overcome steric hindrance. Thedetermination of an appropriate length of linker which allows foroptimal binding and superactivation of the APC or inhibition ofsuperactivation of the APC, is made by screening the ligands withvarying linkers in the superactivation assays detailed in the presentdisclosure.

In other embodiments of the present invention, the multimeric andcomposite supports discussed above have attached multimerized ligands soas to create a “multimerized-multimeric support” and a“multimerized-composite support”, respectively. An embodiment of amultimerized ligand, for example, is obtained by coupling two or morenucleotide sequences encoding the ligand protein or fragments thereof intandem using conventional techniques in molecular biology. Themultimerized form of the ligand is advantageous for many applicationsbecause of the ability to obtain an agent with a better ability tosuperactivate an APC or prevent the superactivation of an APC. Theincorporation of linkers or spacers, such as flexible 8 linkers, betweenthe protein domains which make-up the multimerized agent is alsoadvantageous for some embodiments. The insertion of 8 linkers of anappropriate length between protein binding domains, for example,encourages greater flexibility in the molecule and overcomes sterichindrance between the several proteins. Similarly, the insertion oflinkers between the multimerized ligand and the support encouragesgreater flexibility and reduces steric hindrance presented by thesupport. The determination of an appropriate length of linker whichallows for optimal binding and superactivation of the APC or inhibitionof superactivation of the APC, is made by screening the ligands withvarying linkers in the superactivation assays detailed in thisdisclosure.

In desirable embodiments, the various types of supports discussed aboveare created using proteins or fragments thereof which interact directlyor indirectly with a CD40 molecule of an APC. In preferred embodiments,for example, the multimeric supports, composite supports,multimerized-multimeric supports, or multimerized-composite supports,collectively referred to as “support-bound agents”, have proteins whichcomprise domains found in anti-CD40 antibodies such as FGK45, HM40-3,3/23 5C3, Mab-89, BE-1, EA5, and M3. Additional embodiments includesupport-bound agents having proteins which comprise domains found in theanti-B7.1 (17A10), anti-B7.2 (2D10), CTLA-4-Ig antibodies, MR1antibodies and antibodies which recognize CD40 ligands. In oneembodiment, a composite support comprises both anti-B7.1 (17A10) andanti-B7.2 (2D10) antibodies so as to create a support-bound agent whicheffectively blocks APC superactivation and, concomitantly, activation ofkiller T cells.

In the discussion below, we describe several embodiments of the presentinvention which have therapeutic and/or prophylactic application.

Therapeutic and Prophylactic Embodiments

In the therapeutic and prophylactic embodiments of the presentinvention, the ligands identified as superactivating an APC or blocking,inhibiting, or preventing the activation of a killer T cell(collectively referred to as “superactivation modulating agents”) areincorporated into a pharmaceutical product and are administered to asubject in need. One contemplated method of making a pharmaceuticalinvolves the selection of a ligand which directly or indirectlyinteracts with CD40 of an APC and thereby superactivates the APC.Preferably, a protein, polypeptide fragment, or peptidomimetic,including but not limited to, FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1,EA5, and M3 or fragments or derivatives thereof is selected and isincorporated into a pharmaceutical by conventional techniques.Desirably, the ligand which is incorporated into the pharmaceuticalinteracts with a CD40 molecule of a dendritic cell. Some pharmaceuticalsof the present invention are formulated with an adjuvant and others arefree. Desirable embodiments also comprise the ligand in a support-boundform. Optionally, the superactivation modulating agents are provided inan aggregated form as created, for example, by heating.

In another method, a ligand selected for its ability to block, inhibit,or prevent the activation of a killer T cell by virtue of an interactionwith an APC is incorporated into a pharmaceutical. Accordingly, a ligandwhich interacts with an APC, desirably a dendritic cell, and therebyblocks, inhibits, or prevents superactivation of the APC or activationof a T cell is selected and incorporated into a pharmaceutical byconventional techniques. Preferable ligands include, but are not limitedto, 17A10, 2D10, CTLA-4-Ig, MR1, CD40 5C3, Mab-89, BE-1, EA5, and M3 andfragments or derivatives thereof. Other embodiments include a soluble,non-aggregated form of anti-CD40 or of anti-CD40 ligand. Further,ligands which interact with CD40 by, for example, competitivelyinhibiting the binding of molecules which superactivate an APC throughCD40 are embodiments. A novel class of inhibitors which bind the CD40receptor or its ligand with high avidity but fail to superactivate theAPC are designed using approaches in rational drug design, describedabove. Some pharmaceuticals are formulated in adjuvant and others arefree or are provided in the form of a support-bound agent. As above, anaggregated form of this aspect is created by heating the proteins and isadministered to subjects in need.

Although there may be many other methods of obtaining a pharmaceuticalcomprising a ligand which interacts with a CD40 molecule of an APC andthereby superactivates the APC or which interacts with an APC andthereby blocks, inhibits, or prevents activation of a killer T cell, wecontemplate that a pharmaceutical obtainable by the methods describedabove are within the scope of embodiments of the present invention.Notably, many routes may be taken to arrive at the pharmaceuticalproduct of the processes described above, however, the products remainidentical or equivalent in so far as their ability to superactivate anAPC by interacting with a CD40 molecule or block, inhibit, or preventactivation of a killer T cell by interacting with an APC.

The pharmaceutical products obtainable by the methods detailed in thisdislcosure are useful for the treatment and prevention of cancer,infections, and autoimmune responses. By one approach the ligandsidentified as potentiating a superactivating state in an APC areselected as a cancer or anti-pathogen vaccine component. Additionally,some embodiments of cancer and anti-pathogen vaccine components compriseligands identified for their ability to inhibit the superactivation ofan APC. One method of making a cancer or anti-pathogen vaccine componentincludes contacting an APC, preferably a dendritic cell, with a ligandwhich interacts with the APC and thereby superactivates the APC. Theactivation state of several ligands are analyzed using the approachesdetailed above and the superactivating ligands are incorporated into apharmaceuticals as vaccine components. Preferable components for acancer or anti-pathogen vaccine include, but are not limited to, thesoluble, non-aggregated forms of antibodies which directly or indirectlyinteract with CD40, such as FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5,and M3 and fragments or derivatives thereof and antibodies whichinteract with a CD40 ligand. In some embodiments, vaccine components areformulated in adjuvant and, in others, the vaccine components are free.Desirably, the vaccine components are provided in the form of asupport-bound agent.

The manufacture of a component for immunosuppression is anotherembodiment of the present invention. By one method, an APC, preferably adendritic cell, is contacted with a ligand which interacts with the APCand the activation state of the APC is analyzed by the superactivationassays presented above. Ligands for use as the component for animmunosuppression agent are then selected based on the ability of theligand to block, inhibit, or prevent killer T cell activation byinteracting with the APC. Selected ligands are incorporated intopharmaceuticals and are administered to subjects in need. Preferablecomponents for immunosuppression agents include, but are not limited to,the soluble, non-aggregated forms of antibodies against a CD40 ligandplus 17A10, 2D10, CTLA-4-Ig, and MR1 and fragments or derivativesthereof. In some embodiments, the immunosuppression component isformulated in adjuvant and in other embodiments the immunosuppressioncomponent is free. Desirably, the immunosuppression component isprovided in the form of a support-bound agent.

Additionally, therapeutic and prophylactic agents which comprisesuperactivated APCs are embodiments of the present invention. In thisaspect, for example, an APC, preferably a dendritic cell, is obtainedfrom a subject in need of treatment or prophylaxis for cancer orinfection by a pathogen. The APC is contacted with a cancer cell antigenand the contact results in the formation of an antigen-APC complex. Theantigen-APC complex is then contacted with a ligand which superactivatesthe APC resulting in the formation of a superactivated antigen-APCcomplex. A therapeutically effective amount of the superactivatedantigen-APC complex is administered to the subject so as to treat orprevent cancer or infection by a pathogen. Preferable superactivatingligands include, but are not limited to, FGK45, HM40-3, 3/23, 5C3,Mab-89, BE-1, EA5, and M3 and fragments or derivatives thereof, as wellas, antibodies which interact with a CD40 ligand. Methods ofimmunotherapy of cancer comprising administration of dendritic cells areknown in the art and these techniques are readily adapted for thedelivery of superactivated dendritic cells to treat cancer or infectionby a pathogen given the present disclosure. (See e.g., Gilboa et al.,Cancer Immunol. And Immunoth. 46:82-87 (1998), herein incorporated byreference).

The pharmacologically active compounds of this invention are processedin accordance with conventional methods of galenic pharmacy to producemedicinal agents for administration to subjects, e.g., vertebratesincluding humans. The superactivation modulating agents are incorporatedinto pharmaceutical products with and/or without modification. Further,pharmaceuticals and/or therapeutic agents which deliver thesuperactivation modulating agent or a nucleic acid sequence encoding thesuperactivation modulating agent by several routes are manufactures ofthe present invention. For example, and not by way of limitation, someembodiments use of DNA, RNA, and viral vectors having sequence encodingthe superactivation modulating agent. Nucleic acids encoding a desiredsuperactivation modulating agent are administered alone or incombination with peptides or the ligand protein.

In some aspects, the compounds of this invention are employed inadmixture with conventional excipients, i.e., pharmaceuticallyacceptable organic or inorganic carrier substances suitable forparenteral, enteral (e.g., oral) or topical application which do notdeleteriously react with the active compounds. Suitable pharmaceuticallyacceptable carriers include, but are not limited to, water, saltsolutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,polyetylene glycols, gelatine, carbohydrates such as lactose, amylose orstarch, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritolfatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.The pharmaceutical preparations are, preferably sterilized and ifdesired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or aromatic substances andthe like which do not deleteriously react with the active compounds.

In the following disclosure, doses and methods of administration of asuperactivation modulating agent are provided.

Dosage and Methods of Administration

The effective dose and method of administration of a particularformulation of a superactivation modulating agent varies based on theindividual subject and the stage of the disease, as well as otherfactors known to those of skill in the art. Therapeutic efficacy andtoxicity of such compounds is determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage of such compounds lies preferably within a rangeof circulating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the superactivation modulating agent or to maintainthe state of superactivation of an APC or inhibition of an APC.Additional factors which may be taken into account include the severityof the disease state, age, weight, and gender of the patient; diet, timeand frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions are administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts of superactivation modulating agent vary from 0.1to 100,000 micrograms, up to a total dose of about 10 grams, dependingupon the route of administration. Guidance as to particular dosages andmethods of delivery is provided in the literature. (See U.S. Pat. No.4,657,760; 5,206,344; or 5,225,212.) More specifically, the dosage ofthe superactivation modulating agent of the present invention is onethat provides sufficient agent to attain superactivation of an APC orinhibition of superactivation of an APC. A constant infusion of asuperactivation modulating agent is also provided in some embodiments soas to maintain a stable concentration in the tissues as measured byblood levels.

Routes of administration of the superactivation modulating agentsinclude, but are not limited to, topical, transdermal, parenteral,gastrointestinal, transbronchial, and transalveolar. Topicaladministration is accomplished via a topically applied cream, gel,rinse, etc. containing a peptide agent. Transdermal administration isaccomplished by application of a cream, rinse, gel, etc. capable ofallowing the peptide agent to penetrate the skin and enter the bloodstream. Parenteral routes of administration include, but are not limitedto, electrical or direct injection such as direct injection into acentral venous line, intravenous, intramuscular, intraperitoneal orsubcutaneous injection. Gastrointestinal routes of administrationinclude, but are not limited to, ingestion and rectal. Transbronchialand transalveolar routes of administration include, but are not limitedto, inhalation, either via the mouth or intranasally.

Compositions of superactivation modulating agent-containing compoundssuitable for topical application include, but not limited to,physiologically acceptable implants, ointments, creams, rinses, andgels. Any liquid, gel, or solid, pharmaceutically acceptable base inwhich the superactivation modulating agents are at least minimallysoluble is suitable for topical use in aspects of the present invention.For topical application, there are also employed as non-sprayable forms,viscous to semi-solid or solid forms comprising a carrier compatiblewith topical application and having a dynamic viscosity preferablygreater than water. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders,liniments, salves, aerosols, etc., which are, if desired, sterilized ormixed with auxiliary agents, e.g., preservatives, stabilizers, wettingagents, buffers or salts for influencing osmotic pressure, etc. Fortopical application, also suitable are sprayable aerosol preparationswherein the active ingredient, preferably in combination with a solid orliquid inert carrier material, is packaged in a squeeze bottle or inadmixture with a pressurized volatile, normally gaseous propellant,e.g., a freon.

Compositions of the superactivation modulating agents suitable fortransdermal administration include, but are not limited to,pharmaceutically acceptable suspensions, oils, creams, and ointmentsapplied directly to the skin or incorporated into a protective carriersuch as a transdermal device (“transdermal patch”). Examples of suitablecreams, ointments, etc. can be found, for instance, in the Physician'sDesk Reference. Examples of suitable transdermal devices are described,for instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen,et al.

Compositions of the superactivation modulating agents suitable forparenteral administration include, but are not limited to,pharmaceutically acceptable sterile isotonic solutions. Such solutionsinclude, but are not limited to, saline and phosphate buffered salinefor injection into a central venous line, intravenous, intramuscular,intraperitoneal, or subcutaneous injection of the peptides. Additionalembodiments for parenteral application include injectable, sterile, oilysolutions, suspensions, emulsions, or implants, including suppositories.Ampoules are convenient unit dosages.

Compositions of the superactivation modulating agents suitable fortransbronchial and transalveolar administration include, but not limitedto, various types of aerosols for inhalation. Devices suitable fortransbronchial and transalveolar administration of the superactivationmodulating agents are also embodiments. Such devices include, but arenot limited to, atomizers and vaporizers. Many forms of currentlyavailable atomizers and vaporizers can be readily adapted to deliversuperactivation modulating agents.

Compositions of the superactivation modulating agents suitable forgastrointestinal administration include, but not limited to,pharmaceutically acceptable powders, tablets, pills, dragees, capsules,drops, or liquids for ingestion and suppositories for rectaladministration. A syrup, elixir, or the like can be used wherein asweetened vehicle is employed.

Sustained, pro-drugs, or directed release compositions are alsoformulations used in embodiments of the present invention, e.g.,liposomes or those wherein the active compound is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. It is also possible to freeze-dry the ligandsand use the lyophilizates obtained, for example, for the preparation ofproducts for injection.

Aspects of the invention also include a coating for medical equipment.For example, in some embodiments the superactivation modulating agentsare impregnated into a polymeric medical device such as catheters,stents and prosthetics. Coatings suitable for use in medical devices areprovided by a powder containing the superactivation modulating agents orby polymeric coating into which the superactivation modulating agentsare suspended. Suitable polymeric materials for coatings or devices arethose which are physiologically acceptable and through which atherapeutically effective amount of a superactivation modulating agentcan diffuse. Suitable polymers include, but are not limited to,polyurethane, polymethacrylate, polyamide, polyester, polyethylene,polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl-chloride,cellulose acetate, silicone elastomers, collagen, silk, etc. Suchcoatings are described, for instance, in U.S. Pat. No. 4,612,337, issuedSep. 16, 1986 to Fox et al. which is incorporated herein by reference.

It will be appreciated that the actual preferred amounts of activecompound in a specific case will vary according to the specific compoundbeing utilized, the particular compositions formulated, the mode ofapplication, and the particular situs and organism being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject compounds and of a known agent, e.g., by meansof an appropriate, conventional pharmacological protocol.

The example below discloses the materials and methods used to performthe experiments described above. The materials and methods and theexperiments presented above are also detailed in Ridge et al., Nature393: 474 (1998), herein incorporated by reference.

EXAMPLE 1

Mice and Immunizations:

C57Bl/6 (B6) female and male mice were purchased from Taconic farmsGermantown N.Y. MHC class II knockout mice (KO) were obtained from theNIAID mouse breeding contract (Taconic Farms Inc. Germantown N.Y.). TheKO mice were bred onto both the C57Bl/6 and C57Bl/10 background to N13and N11 respectively. Both types of mice were used in this study. Themice were left unprimed or were primed to the male antigen, H-Y, by anintraperitoneal injection of 3×10⁶ male spleen cells in 200 μl ofsterile phosphate buffered saline (PBS). Mice primed with dendriticcells (see below) were given 5×10⁵ cells in 200 μl PBS per mouseintraperitoneally. Some mice received a second injection of the originalstimulating cells two weeks after the first injection.

CD4 Purification:

Two weeks to 1 year after in vivo priming, spleens were removed from theresponders and depleted for CD4 T cells using the Midi MACs system(Miltenyi Biotec, Germany) with MACS magnetic beads conjugated to theanti-mouse CD4 monoclonal antibody, GK1.5. By FACS analysis with thenon-competing anti-CD4 antibody RM4-4 (Pharmingen, San Diego, Calif.),the depleted populations routinely contained less than 0.2% CD4 cells.

Dendritic Cells:

The enriched dendritic cell preparations were obtained by incubating B6male spleen cells in Iscove's Modified Dulbecco's Medium (IMDM) plus 10%fetal calf serum, 5×10⁻⁵ M 2-mercaptoethanol, glutamine, penn-strep andgentomycin (complete medium) in Falcon tissue culture dishes # 3025 for2 hours at 37° C., removing nonadherent cells and incubating theremaining adherent cells overnight at 37° C. in medium containing 2ng/ml mouse recombinant granulocyte-macrophage colony stimulating factor(rGM-CSF) and 200 U/ml recombinant mouse 1L-4 (Pharmigen). Nonadherentcells were then harvested and further purified over a 50% Percolldensity gradient.

CD40 Cross-Linking:

The enriched dendritic cells were incubated in PBS plus 10% mouse serumfor 10 min on ice, then washed once with cold PBS. They were thenincubated for 20 mins on ice, mixing occasionally, with a Hamster antimouse CD40 (HM40-3) or IgG2a Rat anti mouse CD40 (3/23) (Pharmigan)monoclonal antibodies at 5 μg/ml and 3.5 μg/ml respectively, and washedonce in PBS. The cells were then incubated with either Goat anti Hamsteror Goat anti Rat antibodies (Caltag, Burlingame Calif.) in completemedium containing 2 ng/ml GM-CSF and 200 U/ml IL4 and incubatedovernight at 37° C. The cells were harvested the next day and eitherused as irradiated in vitro stimulators (1500 Rads) or as cells for invivo priming. The dendritic cells used in vivo were washed 3 times insterile PBS before injection.

Infection with Influenza:

Dendritic cells were infected with Influenza virus as described. (54).Briefly 10×10⁶ or less male B6 or KO dendritic cells were resuspended inserum free medium with 1000 HAU of purified Influenza A/PR/8 for 90 minsat 37° C., mixing occasionally, then washed three times in completemedium. These cells were then irradiated (1500 Rads) and used as invitro stimulators.

Stimulating with Marilyn:

B6 dendritic cells were activated by incubating with Marilyn, a CD4 TH1clone specific for H-Y and restricted by Ab that was isolated from aB6×CBA/N female mouse (MHC bxk). 1×10⁶ male dendritic cells wereincubated overnight with 1.5×10⁵ Marilyn. In some experiments, themixture of dendritic cells plus Marilyn was then irradiated 1500 Radsand used as in vitro stimulators. In others, we removed the Marilyns, byFACS sorting with an antibody against H-2^(k), before irradiating andadding the dendritic cells to the cultures. We tested for the efficiencyof T cell removal by staining for CD4, Thy1 and TCR, and by culturing analiquot of the (unirradiated) dendritic cell populations and testing forproliferation. In every case, we found no evidence of contaminatingMarilyn cells.

In Vitro Cultures:

Two weeks or later after the last in vivo immunization, 4×10⁶ spleencells were restimulated in 2 ml cultures with 2×10⁶ irradiated B6 malespleen cells or 1.5×10⁵ of the various populations of dendritic cells,with or with out an exogenous source of mouse IL-2, (10% Rat Con Asupernatant, from which the ConA has been removed, CollaborativeBiomedical Products, Bedford Ma.). Six days later the cultures weretested for their ability to kill male and female targets using the JAMTest. (55).

Antibody Blocking Cultures:

Soluble antibody was titrated 1/2 starting at 500 or 300:g/ml in 100 ulvolumes using a 96 well round bottomed plate. 50 μl containing 1×10⁵unseparated or CD4 depleted spleen cells from a primed B6 female wereadded plus 50:1 containing 5×10⁴ B6 (FIG. 4A) or MHC class II KO (FIG.4B) male dendritic cells superactivated by CD40 crosslinking, to give afinal volume of 200 μl in complete medium. Antibodies reported areHamster anti-mouse B7.1 (17A10), Rat IgG2b anti-mouse B7.2, (2D10),recombinant mouse CTLA4/immunoglobulin fusion protein (CTLA4-Ig), andcontrol antibodies Rat IgG2b anti-mouse Ly5.2 (A20.1.7) used in FIG. 4with the B6 stimulators), and Rat Ig2b anti mouse MHC II (M5/114, usedin FIG. 4 with the MHC KO stimulators). We also used Hamster antibodyUC3 and human recombinant mouse CTLA4/immunoglobulin fusion protein (huCTLA4-Ig) but they did not block and are not reported for the sake ofclarity.

Although the invention has been described with reference to embodimentsand examples, it should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims. All referencescited herein are hereby expressly incorporated by reference.

1. A method of making a component of a pharmaceutical comprising:providing an APC; providing a ligand which interacts with the APC;contacting the APC with the ligand; analyzing the activation state ofthe APC; and selecting the ligand as the component of a pharmaceuticalwherein the ligand superactivates the APC, as determined by the capacityto activate a killer T cell in the absence of a helper T cell.
 2. Themethod of claim 1, wherein the ligand is an antibody selected from thegroup consisting of FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, and M3.3. The method of claim 1, wherein the pharmaceutical is formulated inadjuvant or free.
 4. The method of claim 1, wherein the APC is adendritic cell.
 5. The method of claim 1, wherein the ligand is providedin the form of a support-bound agent.
 6. A method of making a componentof a pharmaceutical comprising: providing an APC; providing a ligandwhich interacts with the APC; contacting the APC with the ligand;analyzing the activation state of the APC; and selecting the ligand asthe component of a pharmaceutical wherein the ligand inhibitssuperacetivating the APC, as determined by the capacity to blockactivating a killer T cell in the absence of a helper T cell.
 7. Themethod of claim 6, wherein the ligand is an antibody selected from thegroup consisting of 17A10, 2D10, CTLA-4-Ig, and MR1.
 8. The method ofclaim 6, wherein the pharmaceutical is formulated in adjuvant or free.9. The method of claim 6, wherein the APC is a dendritic cell.
 10. Themethod of claim 6, wherein the ligand is provided in the form of asupport-bound agent.
 11. A method of treating or preventing cancercomprising the step of administering a therapeutically effective amountof the pharmaceutical of claim 1 to a patient in need thereof.
 12. Amethod of treating or preventing an infection comprising the step ofadministering a therapeutically effective amount of the pharmaceuticalof claim 1 to a patient in need thereof.
 13. Use of the pharmaceuticalof claim 1 for the manufacture of a medicament for the treatment orprevention of cancer.
 14. Use of the pharmaceutical of claim 1 for themanufacture of a medicament for treatment or prevention of infection.15. A method of making a cancer vaccine component comprising: (a)providing an antigen presenting cell (APC) having a tumor antigen; (b)providing a ligand which interacts with the APC; (c) contacting the APCwith the ligand; (d) analyzing the activation state of the APC; and (e)selecting the ligand as the cancer vaccine component wherein the ligandsuperactivates the APC, as determined by the capacity to activate akiller T cell in the absence of a helper T cell.
 16. The method of claim15, wherein the ligand is an antibody selected from the group consistingof FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, and M3.
 17. The methodof claim 15, wherein the cancer vaccine component is formulated inadjuvant or free.
 18. The method of claim 15, wherein the APC is adendritic cell.
 19. The method of claim 15, wherein the ligand isprovided in the form of a support-bound agent.
 20. A method of treatingor preventing cancer comprising the step of administering atherapeutically effective amount of the cancer vaccine component ofclaim 15 to a patient in need thereof.
 21. Use of the cancer vaccinecomponent of claim 15 for the manufacture of a medicament for thetreatment or prevention of cancer.
 22. A method of making ananti-pathogen vaccine component comprising: (a) providing an antigenpresenting cell (APC); (b) providing a ligand which interacts with theAPC; (c) contacting the APC with the ligand; (d) analyzing theactivation state of the APC; and (e) selecting the ligand as theanti-pathogen vaccine component wherein the ligand superactivates theAPC, as determined by the capacity to activate a killer T cell in theabsence of a helper T cell.
 23. The method of claim 22, wherein theligand is an antibody selected from the group consisting of FGK45,HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, and M3.
 24. The method of claim22, wherein the anti-pathogen vaccine component is formulated inadjuvant or free.
 25. The method of claim 22, wherein the APC is adendritic cell.
 26. The method of claim 22, wherein the ligand isprovided in the form of a support-bound agent.
 27. A method of treatingor preventing an infection comprising the step of administering atherapeutically effective amount of the anti-pathogen vaccine componentof claim 22 to a patient in need thereof.
 28. Use of the anti-pathogenvaccine component of claim 22 for the manufacture of a medicament fortreatment or prevention of infection.
 29. A method of making a componentfor immunosupression comprising: (a) providing an antigen presentingcell (APC); (b) providing a ligand which interacts with the APC; (c)contacting the APC with the ligand; (d) analyzing the activation stateof the APC; and (e) selecting the ligand as the component forimmunosupression, wherein the ligand inhibits superactivating the APC,as determined by the capacity to block activating a killer T cell in theabsence of a helper T cell.
 30. The method of claim 22, wherein theligand is an antibody selected from the group consisting of 17A10, 2D10,CTLA-4-Ig, and MR1.
 31. The method of claim 29, wherein the component isformulated in adjuvant or free.
 32. The method of claim 29, wherein theAPC is a dendritic cell.
 33. The method of claim 29, wherein the ligandis provided in the form of a support-bound agent.
 34. A method ofactivating a killer T cell comprising: providing an APC; providing aligand which interacts with the APC; contacting the ligand with the APC,analyzing the activation state of the APC; selecting a superactivatedAPC; and contacting the superactivated APC with a killer T cell in theabsence of a helper T cell resulting in activation of the killer T cell.35. The method of claim 34, wherein the ligand is an antibody selectedfrom the group consisting of FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1,EA5, and M3.
 36. The method of claim 34, wherein the APC is a dendriticcell.
 37. A method of identifying immune responsiveness of a subjectcomprising: (a) providing an APC from the subject; (b) providing aligand which interacts with the APC; (c) contacting the APC with theligand; (d) analyzing the activation state of the APC; and (e) measuringimmune responsiveness, wherein the presence of superactivation indicatesa good immune responsiveness and wherein the absence of superactivationindicates a poor immune responsiveness.
 38. The method of claim 37,wherein the ligand is an antibody selected from the group consisting ofFGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, M3, 17A10, 2D10, CTLA-4-Ig,and MR1.
 39. The method of claim 37, wherein the APC is a dendriticcell.
 40. A kit to identify immune responsiveness comprising: an APC; aligand which interacts with the APC; a means for contacting the APC withthe ligand; a means for analyzing the activation state of the APC; and ameans for measuring immune responsiveness, wherein the presence ofsuperactivation indicates a good immune responsiveness and wherein theabsecnce of superactivation indicates a poor immune responsiveness. 41.The kit of claim 40, wherein the ligand is an antibody selected from thegroup consisting of FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, M3,17A10, 2D10, CTLA-4-Ig, and MR1.
 42. The kit of claim 40, wherein theAPC is a dendritic cell.
 43. A method of treating or preventing cancerin a subject comprising: removing an APC from a subject in need of asuperactivated APC; contacting the APC with a cancer cell antigen so asto form an antigen-APC complex; contacting the antigen-APC complex witha ligand, wherein the ligand superactivates the antigen-APC complex soas to form a superactivated antigen-APC complex; and administering tothe subject a therapeutically effective amount of the superactivatedantigen-APC complex.
 44. The method of claim 43, wherein the ligand isan antibody selected from the group consisting of FGK45, HM40-3, 3/23,5C3, Mab-89, BE-1, EA5, and M3.
 45. A method of treating or preventinginfection by a pathogen in a subject comprising: removing an APC from asubject in need of a superactivated APC; contacting the APC with anantigen from the pathogen so as to form an antigen-APC complex;contacting the antigen-APC complex with a ligand, wherein the ligandsuperactivates the antigen-APC complex so as to form a superactivatedantigen-APC complex; and administering to the subject a therapeuticallyeffective amount of the superactivated antigen-APC complex.
 46. Themethod of claim 45, wherein the ligand is an antibody selected from thegroup consisting of FGK45, HM40-3, 3/23, 5C3, Mab-89, BE-1, EA5, and M3.47. An isolated biological complex comprising a superactivated antigenpresenting cell (APC) attached to a killer T cell and not attached to ahelper T cell.
 48. The isolated biological complex of claim 47, whereinthe APC is a dendritic cell.